Role of gene order and autonomous silencing in the regulation of human beta-globin gene switching

Human beta-like globin genes are expressed in the order in which they lie on the chromosome 5'-epsilon-Ggamma- Agamma-delta-beta-3'. This expression pattern is recapitulated during development in transgenic mice bearing human beta-globin locus yeast artificial chromosomes (beta-YACs). Two modalities of globin gene regulation were studied: (1) the role of gene order in globin gene expression, and (2) the characterization of autonomous silencing of the gamma-globin genes. To understand the effect of gene order, beta-YAC transgenic mice were created using YACs bearing a second marked globin gene located at a non-endogenous site. One construct contained a marked beta-globin gene (betam ) located 5' of the Ggamma-globin gene (betam 5' Ggamma beta-YAC); the other contained a marked Agamma-globin gene ( Agammam) located 5' of the epsilon-globin gene (Agammam 5'epsilon beta-YAC). In the betam 5' Ggamma beta-YAC mice, epsilon-globin and gamma-globin gene expression was temporally normal. beta m-globin gene expression was observed throughout development, and wild-type beta-globin gene expression began at day 14 post-conception. In the Agammam 5'epsilon beta-YAC mice, epsilon- and gammawt-globins were not expressed at any stage of development, and the switch to beta-globin expression was delayed. Agammam-globin was expressed throughout development prior to birth at wild-type levels. However, Agamma m-globin expression was silenced in adult mice, even in the absence of the adult beta-globin gene, demonstrating the existence of an autonomous silencer element. A putative silencer region at -730 to -378 relative to the Agamma-globin mRNA CAP site was deleted in the Agammam 5'epsilon beta-YAC (Delta1s Agammam 5'epsilon beta-YAC), and transgenic mice were produced. Of five lines generated, only one contained a full-length beta-YAC. The Agammam-globin gene was expressed in adult blood, indicating that a silencer region had been deleted. Preliminary in vivo footprinting data demonstrated that the protein binding patterns in adult bone marrow and fetal liver did not differ between the Agammawt- and Agammam-globin promoters, suggesting that both promoters are bound by similar transcription factors at this stage. These studies demonstrate that gene order, distance from the LCR, and gene proximal DNA elements play a role in globin gene regulation.